Chimeric cancer models

ABSTRACT

Chimeric nonhuman mammals useful as inducible spontaneous cancer models are disclosed. The nonhuman mammals are obtained by introducing one or more genetically modified embryonic stem (ES) cells into an early stage embryo, and then implanting the manipulated embryo into a surrogate mother. The ES cells contain a recombinant oncogene, and also may contain a genetic mutation that deletes or inactivates a tumor suppressor gene. Models of different types of cancer are produced by introducing different combinations of genetic mutations into the ES cells that are introduced into the early stage embryo.

This application is a national stage filing under 35 U.S.C. § 371 ofInternational Application No. PCT/US2004/028098, filed Aug. 27, 2004,which claims benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 60/499,277, filed Aug. 28, 2003 and U.S. ProvisionalApplication No. 60/518,249, filed Nov. 7, 2003, the disclosures of eachof which are hereby incorporated-by-reference herein in its entirety.

BACKGROUND OF THE INVENTION

Transgenic and knockout technologies have made possible simulation ofhuman genetic mutations in laboratory animals such as mice. However, itis a time-consuming process to generate complex disease modelscontaining multiple genetic mutations due to the need for mating variousanimal strains to obtain the desired allele combinations in one animal.There is a need for rapid production of animals that harbor multiplegenetic mutations in a substantial number of their cells and so as to beprone to diseases such as cancer.

SUMMARY OF THE INVENTION

It has been discovered that it is possible to make more than two geneticalterations in a nonhuman mammalian embryonic stem (ES) cell whilemaintaining the pluripotency of the ES cell. In addition, it has beendiscovered that when embryonic stem cells containing a recombinantoncogene are injected into an early stage embryo, the resulting chimericmammal is a useful in vivo cancer model. Such chimeric animals of theinvention provide certain advantages over conventional transgenicanimals that contain the same genetic modification(s) present in thegenetically modified cells of the chimeric animal.

Based in part on these discoveries, the invention provides a chimericnonhuman mammal, some of whose cells, but not all of whose cells,contain a recombinant oncogene. In some embodiments of the invention,the recombinant oncogene, e.g., an activated oncogene, is operablylinked to an inducible promoter. In some embodiments of the invention,the cells containing a recombinant oncogene also contain a geneticmutation that causes the mammal to have greater susceptibility to cancerthan a mammal not containing the genetic mutation. In preferredembodiments of the invention the nonhuman mammal is a mouse.

Examples of recombinant oncogenes useful in mammals of the inventioninclude HER2, K-RAS, and EGFR. An example of a genetic mutation usefulfor causing the mammal to have an increased susceptibility to cancer isa mutation that deletes or inactivates a tumor suppressor gene. Examplesof tumor suppressor genes that can be deleted or inactivated in mammalsof the invention are Ink4a, P53 and PTEN. In some embodiments of theinvention, the inducible promoter includes a response element whoseactivity depends on a transactivator encoded by a transactivator geneoperably linked to a tissue specific promoter. An example of such aninducible promoter is a TetO (tetracycline operator) promoter.

The invention provides a nonhuman mammalian ES cell containing a genomecomprising a recombinant oncogene operably linked to an induciblepromoter; and a genetic mutation that causes a mammal containing cellsdescended from the ES cell to have greater susceptibility to cancer thana mammal not containing cells descended from the ES cell. In preferredembodiments of the invention, the ES cell is a mouse ES cell.

The invention also provides differentiated cells derived from an ES cellof the invention, and cancer cells derived from an ES cell of theinvention.

The invention provides a method for obtaining a chimeric nonhumanmammal, some of whose cells, but not all of whose cells, contain agenome comprising: (a) a recombinant oncogene operably linked to aninducible promoter; and (b) a genetic mutation that causes the mammal tohave greater susceptibility to cancer than a mammal not containing thegenetic mutation. The method includes: (a) providing a nonhumanmammalian ES cell containing a genome comprising a recombinant oncogeneoperably linked to an inducible promoter; and a genetic mutation thatcauses a mammal containing cells descended from the ES cell to havegreater susceptibility to cancer than a mammal not containing cellsdescended from the ES cell; (b) introducing the ES cell into a hostnonhuman mammalian embryo, e.g., by injection, thereby producing amanipulated embryo, and (c) implanting the manipulated embryo into asurrogate mother.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. The materials, methods, and examples are illustrative only andnot intended to be limiting. Throughout this specification and claims,the word “comprise,” or variations such as “comprises” or “comprising”will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers. Other features and advantages of the invention are describedin the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of data showing the regression of two tumors from abreast Her2 model mouse (mouse #259) after doxycycline withdrawal fromthe drinking water. Squares represent the tumor that arose in the leftfourth mammary gland. Circles represent the tumor that arose in theright fourth mammary gland. The mouse had been on doxycycline for 6weeks. Day 1 represents the first measurement of the tumors. Aftermeasuring the tumor size, doxycycline was withdrawn from the drinkingwater. Tumors were measured every day for two weeks. Doxycyclinewithdrawal resulted in complete regression of the tumors.

FIG. 2 is a graph of data showing the regression of two tumors from abreast Her2 model mouse (mouse #331) after doxycycline withdrawal fromthe drinking water. Squares represent the tumor that arose in the rightfourth mammary gland. Circles represent the tumor that arose in the leftfourth mammary gland. The mouse was on doxycycline for 7 weeks. Day 1represents the first measurement of the tumors. On day 7, aftermeasuring the tumor size, doxycycline was withdrawn from the water.Tumors were measured every day for 23 days. Doxycycline withdrawalresulted in the complete regression of one tumors and the near completeregression of the other tumor during the study period.

DETAILED DESCRIPTION OF THE INVENTION

This invention features methods of studying the role of a given proteinin disease development (e.g., tumor development), the context-dependentoncogenicity of a genetic mutation, the toxicity of a given protein inorgan development (including survival). The invention also featurescells useful in these methods, e.g., ES cells having more than two(e.g., three, four, five, six, seven or eight) recombinant (i.e., notnaturally occurring) genetic alterations in their genomes. Thisinvention also features a chimeric nonhuman mammal some of whose cellsdiffer genetically from other cells in the mammal and derive from suchES cells. This animal is produced from a multicellular, early-stageembryo, e.g., a blastocyst, into which the ES cells are injected. Inpreferred embodiments, the chimeric animal is disease-prone and developsa disease such as cancer. When the ES cells and the blastocyst intowhich they are injected are derived from the same animal strain, thechimeric mammal is also called a mosaic mammal. In addition to the aboveES cell injection method, a mosaic animal also can be developed from anembryo that has been infected with viral constructs containing thedesired genetic elements.

While preserving the same genetic design as a germline transgenic model,the chimeric model of this invention provides new advantages forstudying diseases involving multiple genes. The chimeric model hassignificantly improved the speed and flexibility of disease modeldevelopment. For example, to generate a transgenic melanoma modeldescribed in Chin et al., Nature 400:468-472 (1999), one would have tobreed three animal lines with four respective genetic alterations—ahomozygous INK4a null mutation (i.e., null mutations on both INK4aalleles), a Tyr-rtTA transgene, and a tetO-H-ras transgene—to obtain atransgenic animal with all four genetic alterations. This requires alarge amount of time. In contrast, a chimeric melanoma model of thisinvention requires no breeding. One only needs to establish ES cellswith all four genetic alterations and inject them into a blastocyst,which develops into an intact animal upon transplantation into theuterus of a surrogate mother. The average time saved can be as much asone year. To establish animal models for a different disease, one needsonly to introduce into ES cells different sets or combinations ofgenetic mutations.

The chimeric model of this invention further allows the study of genesimportant in early development, because the chimeric model providesqualitative correlation between the degree of chimerism and animalviability. The chimeric model also provides a vehicle for testinganti-cancer therapeutics.

The present invention has overcome a major hurdle in ES cell technology.Before this invention, it was widely believed that ES cells subjected tomore than two genetic alterations through recombinant DNA technologywere prone to differentiation and loss of pluripotency.

I. Genetically altered ES cells

An ES cell line of this invention contains more than two recombinantgenetic alterations in its genome. The ES cell line can be establishedby introducing more than two nucleic acid constructs into an ES cellconcurrently or sequentially, where each construct may contain one ormore genetic elements that will cause genetic alterations of the hostgenome. These genetic elements can also be inserted into one singlevector, e.g., a BAC, PAC, YAC or MAC vector.

Exemplary genetic elements include oncogenes, RNA interferenceconstructs, selectable marker genes (e.g., drug selection marker genesand genes encoding fluorescent or luminescent proteins), and knockoutconstructs targeting an endogenous disease-preventing gene (e.g., tumorsuppressor genes). A desired genetic element can be incorporated intothe genome randomly or at a targeted location.

Targeted genetic alterations can introduce a desired change to aspecific location in an endogenous gene involved in a genetic disease,such as neurodegenerative diseases (e.g., the APP gene in Alzheimer'sdisease and the SOD-1 gene in Lou Gehrig's disease), heart diseases(e.g., the TBX-1 or -5 gene in heart diseases and Velo-Cardio-FacialSyndrome), diabetes (e.g., the AKT gene in Type II), and autoimmunediseases. Examples of the changes include a null (knock out) mutation toa tumor suppressor gene or an activating mutation (knock in) to acellular oncogene. For instance, one can replace a coding or regulatoryregion of a tumor suppressor gene with a selectable marker gene flankedby a pair of LoxP sites; or insert a dominant negative mutation into atumor suppressor gene; or replace the native promoter of a cellularoncogene with a constitutive or inducible promoter; or inserting anactivating mutation into a cellular oncogene (see, e.g., Johnson et al.,Nature 410:1111-6 (2001)). Such a genetic alteration can be accomplishedby homologous recombination. In a nucleic acid construct used forhomologous recombination, the genetic alteration to be introduced intothe host genome is flanked by sequences homologous to the targetedgenomic region.

Oncogenes useful in establishing the chimeric disease model of thisinvention include, without limitation, those encoding K-RAS, H-RAS,N-RAS, epidermal growth factor receptor (EGFR), MDM2, TGF-β, RhoC, AKTfamily members, myc (e.g., c-myc), β-catenin, PDGF, C-MET, PI3K-CA,CDK4, cyclin B1, cycline D1, estrogen receptor gene, progesteronereceptor gene, Her2 (also known as neu or ErbB2), other ErbB genes(including ErbB1, ErbB3, and ErbB4), genes in the MAPK and PI3K-AKTsignal transduction pathways, TGFα, ras-GAP, Shc, Nck, Src, Yes, Fyn,Wnt, and Bcl2 anti-apoptotic family members (e.g., Bcl2) as well astheir activated forms, and viral proteins such as PyV MT and SV40 Tantigens. Activating mutations of these oncogenes (e.g., Her2V664E,K-RasG12D, and β-cateninΔ131) can also be used.

Tumor suppressor genes whose inactivation is useful in establishing thechimeric disease model include, without limitation, Rb, P53, INK4a,PTEN, LATS, Apafl, Caspase 8, APC, DPC4, KLF6, GSTP1, ELAC2/HPC2 orNKX3.1. Other examples of tumor suppressor genes are those involved inDNA damage repair (e.g., ATM, CHK2, ATR, BRCA1, BRCA2, MSH2, MSH6, PMS2,Ku70, Ku80, DNA/PK, XRCC4 or MLH1), and cell signaling anddifferentiation (e.g., Neurofibromatosis Type 1, Neurofibromatosis Type2, Adenomatous Polyposis Coli, the Wilms tumor-suppressor protein,Patched or FHIT). In addition to targeted mutation, tumor suppressorgenes (or any other disease-preventing genes) can be inactivated by anantisense RNA, RNA interference (RNAi), or ribozyme agent expressed froma construct stably integrated into the host genome.

In some embodiments of this invention, the chimeric disease model isdeveloped from ES cells that contain an introduced active oncogene aswell as one or more inactivated endogenous tumor suppressor gene(s). Forexample, the ES cells can contain genetic alterations that result in theexpression of an activated form of EGFR (designated as EGFR*) incombination with reduced p16^(INK4a) or p19^(ARF) expression (e.g.,genetic alterations that produce an EGFR*⁺ and INK4a/ARF^(−/−)genotype); genetic alterations that result in PDGF expression incombination with reduced p53 expression (e.g., genetic alterations thatproduce a PDGF⁺ and p53^(−/−) genotype); genetic alterations that resultin TGF-α expression in combination with reduced p53 expression (e.g.,genetic alterations that produce a TGFα⁺ and p53^(−/−) genotype); andgenetic alterations that result in reduced PTEN expression and reducedp16^(INK4a) or p19^(ARF) expression (e.g., genetic alterations thatproduce a PTEN^(−/−) and INK4a/ARF^(−/−) genotype).

An example of suitable set of genetic modifications for production of alung cancer model is TetO-EGFR* CCSP-rtTA, p53^(−/−), TetO-luciferaseand PGK-puromycin (selectable antibiotic resistance marker). An exampleof a suitable set of genetic modifications for production of a coloncancer model is TetO-K-RAS, villin-rtTA, APC^(−/−), TetO-luciferase andPGK-puromycin. An example of a suitable set of genetic modifications forproduction of a glioblastoma cancer model is TetO-EGFR*, Nestin-rtTA,p53^(−/−), TetO-luciferase and PGK-puromycin. An example of a suitableset of genetic modifications for production of a prostate cancer modelis TetO-AKT1, probasin-rtTA, Rb^(−/−), TetO-luciferase andPGK-puromycin. An example of a suitable set of genetic modifications forproduction of a liver cancer model is TetO-β, catenin, ApoE-rtTA,NF1^(−/−), TetO-luciferase and PGK-puromycin.

Various vectors can be used to make the nucleic acid constructs for thisinvention. These vectors can be based on plasmids or viruses such asretroviruses, adenoviruses, and lentiviruses. The vectors can beintroduced into ES cells via a variety of methods, including but notlimited to, cell fusion (e.g., spheroplast fusion), liposome fusion(transposomes), conventional nucleic acid transfection methods (such ascalcium phosphate precipitation, electroporation, microinjection), andinfection by viral vectors. A variety of methods can be used to screenfor ES cells that have stably incorporated the desired geneticalterations. Such methods include, without limitation, detection of drugresistance where a drug selection marker gene (e.g., aneomycin-resistant gene, a puromycin-resistant gene, or ahygromycin-resistant gene) is co-introduced; detection offluorescence/bioluminescence emission where a fluorescent/bioluminescentmarker gene (e.g., a gene encoding a green, yellow, blue or redfluorescent protein, and Luciferase genes) is co-introduced; polymerasechain reaction (“PCR”); and Southern blot analysis.

The ES cells can be genetically altered to contain a nucleic acidsequence that is regulated in an inducible manner. For example, anintroduced oncogene or RNA interference sequence can be placed under thecontrol of an inducible promoter such as the tetracycline-regulatedpromoter system described in e.g., WO 01/09308. In this case,administering the inducing agent (e.g., tetracycline or doxycycline) viafood or drinking water to a chimeric animal, in which at least somecells originate from these genetically altered ES cells, can result inexpression of the oncogene or RNAi product. Other inducible promotersinclude, without limitation, a metallothionine promoter, the IPTG/lacIpromoter system, the ecdysone promoter system, and the “lox stop lox”system for irreversibly deleting inhibitory sequences for translation ortranscription. Instead of inducible promoters, the expression of adisease-causing gene can also be inducibly switched on or off by fusingthe gene's polypeptide product to, e.g., an estrogen receptorpolypeptide sequence, where administration of estrogen or an estrogenanalog (e.g., hydroxytamoxifen) will allow the correct folding of thepolypeptide into a functional protein.

The introduced polypeptide-encoding or interfering RNA-encoding sequencecan also be placed under a general, constitutively active promoter,e.g., a cytomegalovirus (CMV) promoter, EF1α, retroviral LTRs, and SV40early region. Alternatively, the coding sequence can be placed under thecontrol of a tissue-specific promoter, such as a tyrosinase promoter ora TRP2 promoter in the case of melanoma cells and melanocytes; an MMTVor WAP promoter in the case of breast cells and/or cancers; a Villin orFABP promoter in the case of intestinal cells and/or cancers; a PDXpromoter in the case of pancreatic cells; a RIP promoter in the case ofpancreatic beta cells; a Keratin promoter in the case of keratinocytes;a Probasin promoter in the case of prostatic epithelium; a Nestin orGFAP promoter in the case of central nervous system (CNS) cells and/orcancers; a Tyrosine Hydroxylase, S100 promoter or neurofilament promoterin the case of neurons; the pancreas-specific promoter described inEdlund et al. Science 230:912-916 (1985); a Clara cell secretory proteinpromoter in the case of lung cancer; and an Alpha myosin promoter in thecase of cardiac cells.

Developmentally regulated promoters may also be selected. They include,without limitation, the murine hox promoters (Kessel and Gruss, Science249:374-379 (1990)) and the α-fetoprotein promoter (Campes and Tilghman,Genes Dev. 3:537-546 (1989)).

Any ES cell lines that provide adequate chimerism can be used in thisinvention. The cell lines include, without limitation, E14.1, WW6, CCE,J1, and AB1. See also Alex Joyner, Ed., Gene Targeting, A PracticalApproach, Chapter 4 (Virginia Papaioannou), Oxford Press, 2^(nd) Ed.,(2000). In some embodiments, the ES cell lines provide 10% or higherchimerism. In some embodiments, the ES cell lines provide 90% or higherchimerism.

II. Chimeric Nonhuman Animals

As used herein, “chimeric” means chimeric in terms of ontogeny.Accordingly, a chimeric nonhuman mammal is an animal that has grown,i.e., developed, directly from a multicellular embryo into which atleast one genetically modified ES cell has been injected or aggregated.A chimeric nonhuman mammal of the invention is to be distinguished froma morphologically developed animal that has received a xenograft, e.g.,an organ graft, a tissue graft, or a tumor graft from another animal.

As used herein, “nonhuman mammal” means any mammal other than a human,e.g. a rat, a mouse, a hamster or a guinea pig.

A chimeric nonhuman mammal of the invention can be generated byintroducing ES cells containing into a host embryo. This can be done,for example, by blastocyst injection or aggregation with earlier stagepre-implantation embryos (e.g., eight-cell embryo). The embryo issubsequently transferred into a surrogate mother for gestation.Chimerism in the born animal can be determined by phenotype (such as furcolor, if the host embryo and the ES cells are derived from animalstrains of different fur colors), PCR, Southern blot analysis, orbiochemical or molecular analysis of polymorphic genes (such as glucosephosphate isomerase). To facilitate identification of chimeric animalshaving a desired genetic alteration, one can co-introduce a detectablereporter gene and the desired genetic alteration into the ES cells.Exemplary reporter genes include those that encode a fluorescent proteinsuch as a green fluorescent protein, a yellow fluorescent protein, ablue fluorescent protein, or a luminescent protein such as luciferase orβ-galactosidase.

To increase the contribution of introduced ES cells to a specifictissue, one can use a host embryo that is deficient in generating thatissue. This can be accomplished by any suitable method, includinginducible expression of a toxin gene, e.g., diphtheria toxin, in aspecific cell type, or tissue-specific deletion of genes needed forgenerating this cell type. In such a complementation system, all or mostof the cells of the desired cell type or tissue will be derived from theintroduced ES cells.

In some embodiments of the invention, the nonhuman mammals areimmunocompromised or immunodeficient. Diseases may develop sooner and/orfaster in such animals. To develop such animals, one can use blastocystsderived from, for example, an X-linked SCID animal, or a RAG1−/− orRAG2−/− animal.

The chimeric animals of this invention provide efficient models todevelop diseases that originate from introduced ES cells. In aninducible cancer model of this invention, the animal may develop cancerwithin a few months of the induction of oncogene the expression. Theanimal may also be treated with carcinogens, e.g.,9,10-dimethyl-1,2-benzanthracene or ENU, to expedite this process.

The chimeric animal models of this invention provide flexibility indeveloping models of different diseases. For example, ES cell lines maybe established for different cancer models by knocking out a tumorsuppressor gene (e.g., p53) and introducing a reporter gene (e.g.,luciferase), a tissue-specific reverse tetracycline transactivator gene(i.e., MMTV-rtTA) and an oncogene of choice (e.g., Akt, Her2V664E, Her2,Bcl2, K-Ras and Cyclin D1) under the control of a promoter regulated byreverse tetracycline transactivator (rtTA). These cancer models allowthe comparison study of cancers of different etiology, and comparisonstudy of different oncogenes in cancer development.

III. Exemplary Uses

The chimeric animals of this invention and diseased cells derived fromthe animals can be used to delineate the initiation, progression,maintenance, regression, minimal residual disease, recurrence, or anyother developmental stages of a specific disease such as a cancer. Theycan also be used for drug target identification, target validation, andefficacy testing during drug development.

A. Identification of New Cancer Related Genes

The chimeric cancer models of this invention can be used to examine theoncogenicity of any gene, and to identify new cancer related genes. Forinstance, a candidate oncogene and a null mutation of an endogenoustumor suppressor gene (or an RNAi construct targeting the tumorsuppressor gene) may be co-introduced into ES cells. A higher incidencerate, or shorter latency, of cancer originating from the ES cells in aresulting chimeric animal, as compared to that originating from ES cellscontaining only the null mutation in a control animal, indicates thatthe candidate gene is an oncogene.

In addition, a gene expression profile for a chimeric animal havingcancer due to the expression of an introduced oncogene via ES cells canbe established. Then, comparisons of gene expression profiles atdifferent stages of cancer development can be performed to identifygenes whose expression patterns are altered. Techniques used toestablish gene expression profiles include the use of suppressionsubtraction (in cell culture), differential display, proteomic analysis,serial analysis of gene expression (SAGE) and comparative genomichybridization (CGH). To allow high throughput profiling, cDNA and/oroligonucleotide microarrays can be used.

Gene expression profiles in separate animal models that containdifferent genetic alterations predisposing them to the same type ofcancer can be compared to identify tumor-related genes. As discussedbelow, surrogate biomarkers. For instance, overexpression of any one ofAkt, Her2, Bcl-2, K-ras and cycline D1, or activating mutations of anyof these genes (e.g., Her2V664E) can cause breast cancer. By comparinggene expression profiles in breast cancer tissues isolated fromdifferent chimeric animals each containing mutations in one of thesegenes, one can obtain information as to the different pathways involvedin the development (including initiation, maintenance and regression) ofbreast cancer. This information will be valuable in determiningtherapeutic regimen for breast cancer caused by different geneticlesions.

B. Identification of Surrogate Biomarkers

The chimeric animals of the invention also can be used to identifysurrogate biomarkers for diagnosis or to follow disease progression in anonhuman animal (e.g., a mouse, a rat, or a nonhuman primate). Thebiomarkers can be identified based on the differences between theexpression profiles for a chimeric animal that has developed a diseaseand one that has not developed the disease. Blood, urine or other bodyfluids from the animals can be tested with ELISAs or other assays todetermine which biomarkers are released from the diseased tissue intocirculation during genesis, maintenance, progression or regression ofthe disease. Such diagnosis may involve detecting the expression oractivity level of the biomarker, wherein an abnormally high levelrelative to control (e.g., at least about 50%, 100%, 150%, 200%, 250%,or 300% higher) is indicative of an abnormal condition. These biomarkersare particularly useful clinically in following disease progression postdisease therapy. These biomarkers can also be used clinically to assessthe toxicity of any disease therapy.

C. Identification of Therapeutic Agents

The chimeric animals of the invention can be used to screen therapeuticagents to treat a disease. One such method involves administering acandidate compound to an animal that has developed a cancer. Then onecan observe the effect of the compound, if any, on the cancer. One canmeasure, e.g., tumor size, metastasis, or angiogenesis. Alternatively,one can observe the effect of the compound on the expression or activitylevel of a biomarker for the cancer.

D. Identification of Genes Crucial for Organ Survival or Function

To identify a gene crucial for organ survival or function, one canevaluate regeneration in an organ containing cells having a geneticalteration in a candidate gene. A genetic alteration of a candidate genecan be introduced into an ES cell line so that the expression of thecandidate gene is inhibited or stimulated through an induciblemechanism. In a resulting chimeric animal, the expression of thecandidate gene in the chimeric cells may be induced by an inducermolecule or inhibited by RNA interference. Upon induction or inhibition,the degree of chimerism may change in the organ. For example, thepercentage of chimeric cells may change in a liver after induction ofthe expression of a candidate gene. If loss or overexpression of thecandidate gene product is detrimental to liver cell survival, chimericcells will die upon induction, and the chimerism in the liver willdecrease.

E. Study of Differentiation Potential of ES Cells

In some embodiments, one can study differentiation potential,of such EScells in vivo in the context of tumorigenesis. A chimeric animal havingcells that originate from ES cells containing certain geneticalterations may be more prone to develop tumors in certain tissues. Acomparison of tumor development in different tissues of the chimericanimals will thus provide insight into which genetic alterationspreferentially cause tumors in which tissues.

F. Study of Cell/Cell Interactions in Tumor Formation

The chimeric animals of the invention may be used to study thecontribution of cell/cell interaction to tumor formation. The cell/cellinteraction may involve cells having different genetic backgrounds thatoriginate from ES cells having different genetic alterations, or fromcells derived from two different sources, e.g., the introduced ES cellsand the host blastocyst cells.

By way of example, the host blastocyst for ES cell injection isgenetically modified to allow studies of stromal contribution to tumordevelopment. The host blastocyst can be derived from, e.g., a transgenicanimal that produces elevated levels of a growth factor or cytokine in aspecific tissue; thus one can study the effect of this growth factor orcytokine on the development of tumors arising from cellular progeny ofthe introduced ES cells in that tissue. The host blastocyst can also bederived from, e.g., a transgenic animal that is deficient in a certaincellular product (e.g., an adhesion molecule, an angiogenesis factor, areceptor, or a signaling molecule) in a specific tissue; thus one canstudy the role of this cellular product in providing stromal support fortumor development in that tissue.

In another example, ES cells from two ES cell lines having differentgenetic alterations are co-introduced into blastocysts and the resultingchimeric animals may develop a tumor that originates from both ES celllines. Tumor formation in such chimeric animals can be compared withchimeric animals containing tumor cells originating from only one of thetwo ES cell lines. Such a comparison will provide insight into theeffect of interactions between cells having the different geneticalterations on tumor formation.

G. Study of Toxicity of a Genetic Element

The chimeric animals of this invention can be used to measurequantitatively the toxicity of a genetic element, e.g., a geneintroduced into a cell (e.g., an inducible oncogene), an overexpressedendogenous gene, an RNAi construct against an endogenous gene (e.g., atumor suppressor gene), a construct that knocks out an endogenous gene,etc. To do this, one can generate a chimeric animal from a hostblastocyst that has been injected with ES cells containing the testgenetic element, wherein the host blastocyst itself does not containthis test genetic element. He can then compare the chimerism of theanimal with that of a control animal, e.g., a chimeric animal developedwith control ES cells that do not contain the genetic element, or achimeric animal developed with the same ES cells but the test geneticelement in those cells or progeny thereof are not expressed (e.g.,induced). A lower degree of chimerism indicates that the test geneticelement affects development negatively. A higher degree of chimerismindicates the opposite.

A variety of methods can be used to determine the degree of chimerism.For instance, a biopsy of the organ can be obtained and analyzed bybiochemical methods. Alternatively, the host embryo and the ES cell areengineered to express a different fluorescent protein (e.g., one of GFP,RFP, and YFP), and the ratio of the two fluorescent signals, which isindicative of chimerism, is analyzed by in vivo imaging.

H. Mammalian Second Site Suppressor (MaSS) Screen

The chimeric animals of this invention can be used in a MaSS screen toidentify cancer-related genes. In general, a MaSS screen involves: (a)maintaining cells in which tumorigenicity depends on the expression ofan inducible oncogene under conditions in which the expression of theoncogene is not induced; (b) introducing into the cells a nucleic acidmolecule, e.g., a retrovirus, that integrates into the genomes of thecells, thereby tagging the loci at which it integrates; (c) identifyingcells in which tumorigenicity has been induced by integration of thenucleic acid molecule; and (d) identifying genes that have been taggedby the integrated nucleic acid molecule. Methods and materials forperformance of a MaSS screen are described in WO 02/079419.

IV. Examples

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only, and are not to beconstrued as limiting the scope or content of the invention in any way.

Example 1 HER2 Lung Cancer Model

The frequency of HER2/neu overexpression in lung cancer has mostly beenstudied in non-small cell lung cancer, and the reported frequencies ofHER2/neu overexpression range from 5 to 59%. The patients withHER2/neu-positive tumors have significantly shorter survival. HER2/neuoverexpression has also been shown to contribute to tumorigenesis inlung tumor cell lines. See, e.g., Hirsch et al., Lung Cancer 36:263-4(2002); and Gatzemeier et al., Annals of Oncology 15:19-27 (2004).However, anti-HER2/neu antibodies (trastuzumab or HERCEPTIN®) do notseem efficacious in treating HER2-positive non-small cell lung cancer inhumans. Gatzemeier et al., supra.

Chimeric mice that inducibly overexpress HER2/neu in their lungs anddevelop lung cancer shortly after induction were produced. These micewere used to show a cause-effect relationship between HER2/neu and lungcancer. Since a human HER2/neu coding sequence was used in making themice, the mice are useful for, inter alia, developing lung cancertherapeutics that target HER2/neu in human patients and for testing theanti-lung cancer efficacy of known HER2/neu drugs. The mice were made asfollows.

An INK4a−/− ES cell line was first co-transfected with two expressionconstructs. The first construct (CCSP-rtTA) contained a reversetetracycline transactivator (rtTA) coding sequence linked operably to aClara cell secretory protein (CCSP) promoter (Fisher et al., Genes &Development 15:3249-62 (2001)). The second construct (TetO-luc)contained a luciferase (luc) coding sequence linked operably to aminimal-fos promoter containing a tetracycline operator sequence (TetO).ES cell lines containing both CCSP-rtTA and TetO-luc were established byco-transfection of these two constructs together with PGK-puromycin.Puromycin resistant cells were clonally isolated and genotyped by PCRand Southern Blot for the presence of both CCSP-rtTA and TetOLuc.Fifteen of these resultant cell lines were injected into blastocysts togenerate chimeric mice, to assess the relative performance of the celllines. Inducibility of the luciferase reporter gene in the lung wasstudied in vitro by comparison of luminescent signals of lung samplesdissected from chimeras that had or had not been exposed to doxycycline.

Two of the cell lines that passed the inducibility analysis were furthertransfected with a third construct containing a human HER2/neu codingsequence linked operably to TetO. The HER2/neu polypeptide productcontained a V664E/Neu mutation (i.e., substitution of glutamic acid forvaline at position 664) along with PGK-hygromycin 659). Thus, in achimeric mouse containing cells descended from these engineered EScells, the expression of the HER2/neu gene was under the control of thertTA and tetracycline or a tetracycline analog (e.g., doxycycline). Andsince the rtTA was under the control of the lung-specific CCSP promoter,the HER/neu oncogene would be expressed inducibly only in the lungs.

Then, twelve of these ES cell lines were injected into mouse blastocystsfrom C57/BL6 females. The injected blastocysts were transferred tosurrogate mothers for gestation. As assessed qualitatively (by coatcolor), approximately 5% to greater than 90% of the resulting animals'body developed in mosaic fashion from the engineered ES cells. The micewere then given doxycycline-containing drinking water (2 mg/ml) at weekfour. The lung tissues from the chimeric mice were analyzed by RT-PCRand immunohistochemistry. Lung adenomas were observed within seven weeksafter the treatment started. Within two to five months, invasiveadenocarcinoma developed in the lungs. Thus, these data demonstratedthat HER2/neu can initiate lung cancer.

The chimeric HER2/neu mice offered several advantages over conventionaltransgenic lung cancer models containing the same genetic modifications.In transgenic mice that inducibly expressed HER2/neu in the lungs,severe hyperplasia developed throughout the lungs within two to threeweeks of induction. The lungs became nonfunctional, resulting in deathbefore any tumor had a chance to develop. Similarly, in transgenic micethat inducibly expressed K-ras in the lungs (Fisher et al., supra), thetumor loads were so high that the animals died before the tumors had achance to become invasive. In the chimeric HER2/neu mice, in contrast,there was enough healthy lung tissue left to support survival afterhyperplasia developed. As a result, transformed lung cells had time toprogress into more malignant, invasive tumors. Therefore, the chimericmice allowed more detailed studies of tumor development.

Example 2 HER2 Breast Cancer Model

Ink4a homozygous null ES cells were co-transfected with the followingfour constructs, as separate fragments: MMTV-rtTA,TetO-Her2^(V664 Eneu), TetO-luciferase and PGK-puromycin.Puromycin-resistant cells were genotyped by PCR and Southern blot.Inducibility of the oncogenes in ES cells was analyzed by northern blot.The transfected ES cells were injected into C57BL/6 blastocysts, whichwere transplanted into pseudo-pregnant female mice for gestation leadingto birth of the chimeric mice.

The mouse mammary tumor virus long terminal repeat (MMTV) is used todrive breast-specific expression of the reverse tetracyclinetransactivator (rtTA). The rtTA provides for breast-specific expressionof the HER2 activated oncogene when doxycycline is provided to the mice,e.g., in their drinking water.

Inducibility of the HER2 oncogene and luciferase was confirmed by RT-PCRand luciferase assay (respectively), using cultured cells derived fromthe mouse. Mammary glands were removed from chimeric mice and digestedwith collagenase. Half of the organoids collected were cultured in thepresence of doxycycline, and the other half was cultured withoutdoxycycline. After five days in culture, the cells were trypsinized, andone tenth of the cells were used for luciferase assay, and the rest wereused for RNA extraction.

The histology analysis of tumors harvested from HER2 breast cancer modelmice showed invasive adenocarcinomas. Two major patterns weredistinguished. They were a solid sheet growth pattern, and a nestedgrowth pattern with necrotic centers.

Immunohistochemistry analysis of mammary tumors from HER2 breast cancermodel mice revealed two cell types within the tumors. The first celltype was epithelial origin (cytokeratin positive), and showed HER2expression and strong proliferation. The second cell type wasmesenchymal origin with fibroblast-like appearance. These cells werecollagen positive. These cells did not show strong proliferation, andthey displayed stromal function. Apoptosis was seen in the necroticcenters of the epithelial part of the tumors.

Tumor regression studies were performed using the HER2 breast cancermodel mice. Two mice, each carrying more than two doxycycline-inducedtumors, were selected. The tumor size of two tumors each was measuredusing calipers before and after doxycycline was withdrawn from thedrinking water. Doxycycline was withdrawn at day six. Tumor size wasmeasured daily. The tumor size measurements were used to calculate thetumor volume. Results were plotted and the regression of the tumors wasdetermined. All tumors regressed, displaying doxycycline-dependence(FIGS. 1 and 2). Immunohistochemistry analysis of tumor regressionconfirmed doxycycline-dependent HER2 expression. Thus, growth andmaintenance of the tumors were shown to depend on HER2 expression.

Example 3 K-RAS Breast Cancer Model

Ink4a homozygous null ES cells were co-transfected with the followingfour constructs, as separate fragments: MMTV-rtTA, TetO-K-RAS^(G12V),TetO-luciferase and PGK-puromycin. Puromycin-resistant cells weregenotyped by PCR and Southern blot. Inducibility of the oncogenes in EScells was analyzed by northern blot. The transfected ES cells wereinjected into C57BL/6 blastocysts, which were transplanted intopseudo-pregnant female mice for gestation leading to birth of thechimeric mice.

Inducibility of the K-RAS oncogene and luciferase was confirmed byRT-PCR and luciferase assay (respectively), using cultured cells derivedfrom the mouse. Mammary glands were removed from chimeric mice anddigested with collagenase. Half of the organoids collected were culturedin the presence of doxycycline, and the other half was cultured withoutdoxycycline. After five days in culture, the cells were trypsinized, andone tenth of the cells were used for luciferase assay, and the rest wereused for RNA extraction.

The histology analysis of tumors harvested from K-RAS breast cancermodel mice showed hyperplasia in the epithelial ducts of the mammarygland after 2 months on doxycycline. This hyperplasia is observed untilthe end of the observation period (6 month on doxycycline).

Example 4 K-RAS Lung Cancer Model

Ink4a homozygous null ES cells were co-transfected with the followingfour constructs, as separate fragments: CCSP-rtTA, TetO-K-RAS^(G12V),TetO-luciferase and PGK-puromycin. Puromycin-resistant cells weregenotyped by PCR and Southern blot. Inducibility of the oncogenes in EScells was analyzed by northern blot. The transfected ES cells wereinjected into C57BL/6 blastocysts, which were transplanted intopseudo-pregnant female mice for gestation leading to birth of thechimeric mice.

Inducibility of the K-RAS oncogene and luciferase was confirmed byRT-PCR, Northern blot and luciferase assay (respectively), using tissuederived from the mouse lungs. Lungs were removed from chimeric mice andhomogenized. Homogenized material from mice on and off Doxycycline wascompared in Luciferase assays and RNA expression analysis.

The histological analysis of lungs from the mice on doxycycline revealedmultiple adenocarcinomas in the lung. The adenocarcinomas can be seen asearly as 3 month on doxycycline. Chimeras with a low percentage ofES-cells showed a longer latency for tumor development with an overalldecreased tumor burden. The tumors derive from type II pneumocytes asanalyzed by immunohistochemistry using antibodies against the SPC andCC10 antigens.

Other embodiments are within the following claims.

1. A method of making a chimeric mouse cancer model, comprising: (a)providing a mouse ES cell whose genome comprises homozygous inactivationof an endogenous tumor suppressor gene; (b) transfecting the mouse EScell with (i) a first vector comprising a recombinant oncogene operablylinked to a tetracycline dependent inducible promoter, and (ii) a secondvector comprising a tissue- specific reverse tetracycline transactivatorgene; (c) injecting the mouse ES cell into a mouse blastocyst, and (d)transferring the blastocyst into a pseudo-pregnant female mouse forgestation leading to birth of the chimeric mouse; wherein induction ofthe oncogene following administration of tetracycline analog to saidmouse results in the development of tumor.
 2. The method of claim 1,wherein the recombinant oncogene is selected from the group consistingof K-RAS, H-RAS, N-RAS, EGFR, MDM2, RhoC, AKT1, AKT2, c-myc, n-myc,β-catenin, PDGF, C-MET, PI3K-CA, CDK4, cyclin B1, cyclin D1, estrogenreceptor gene, progesterone receptor gene, Her2, ErbB1, ErbB3, ErbB4,TGF-α, TGF-β, ras-GAP, Shc, Nck, Src, Yes, Fyn, Wnt, Bcl₂, PyV MT, andSV40 LT.
 3. The method of claim 2, wherein the recombinant oncogene isHER2, K-RAS or EGFR.
 4. The method of claim 1, wherein the tumorsuppressor gene is selected from the group consisting of Rb, P53, INK4a,PTEN, LATS, Apafl, Caspase 8, APC, DPC4, KLF6, GSTP1, ELAC2/HPC2 orNKX3.1, ATM, CHK2, ATR., BRCAI, BRCA2, MSH2, MSH6, PMS2, Ku70, Ku80,DNA/PK, XRCC4 or MLH 1, NF 1, NF2, APC, Adenomatous Polyposis Coli(APC), Wilms tumor-suppressor protein (WT), Patched and FHIT.
 5. Themethod of claim 4, wherein the tumor suppressor gene is INK4a.
 6. Themethod of claim 1, wherein the inducible promoter is a TetO promoter.